Fully resting CD4+ T cells are non-permissive for HIV-1 replication and only activated cells support high levels of viral replication. In resting cells, viral entry is not limiting for infection, but rather a number of post-entry steps in the replication cycle are defective. These defects are likely to be the result of limiting levels of cellular co-factors. We propose to test the hypothesis that some of these limiting co-factors in resting CD4+ T cells are subject to miRNA repression, and upon T cell activation this repression is relieved, leading to expression of the co-factors and a permissive cellular environment for HIV-1 replication. MiRNAs are short (~22 nucleotide) RNAs that form imperfect duplexes with target mRNAs and repress translation. MiRNAs typically target the 3' UTR of their target mRNAs. Our recent work has provided strong support for the hypothesis that miRNAs in resting CD4+ T cells repress an HIV-1 co-factor known as Cyclin T1. We propose to investigate in detail miRNA repression of Cyclin T1 in resting CD4+ T cells. We also propose to identify other co-factor mRNAs that are subject to miRNA repression in these cells. Additionally, the use of cocaine by HIV-infected individuals is associated with a more rapid progression to AIDS. Cocaine stimulates HIV-1 replication and it has been shown to alter the expression pattern of cellular mRNAs and miRNAs. We will test the hypothesis that cocaine's stimulation of HIV-1 replication involves changes in expression levels of miRNAs of relevance to HIV-1 replication. Our preliminary data with miR-27b and miR-223, miRNAs that can repress Cyclin T1, have provided initial support for this hypothesis. Completion of the proposed research will provide new insight into mechanisms that restrict HIV-1 replication in resting CD4+ T cells, as well as insight into mechanisms whereby cocaine enhances viral replication. This insight should be valuable in designing new therapeutic approaches to treat HIV-1 infection, especially in individuals with a history of cocaine abuse. PUBLIC HEALTH RELEVANCE: We will identify small non-coding RNAs that regulate HIV-1 replication, and we will also determine if cocaine affects the levels of these RNAs. Our research may improve patient treatment and increase understanding about how cocaine use accelerates progress to AIDS.